Achieving an improved understanding of Schwann cell (SC) proliferation and myelination is important for maximizing the clinical promise of these cells in the repair of nervous system injury and disease. Extensive work has shown that neuregulins and cyclic adenosine mono-phosphate (cAMP) are major regulators of both SC proliferation and myelination, but the signaling pathways by which their actions are manifest have received ittle attention. In four Specific Aims, we will test a central hypothesis that cAMP exerts its effects on SC function by regulating intracellular signaling initiated by growth factors, including neuregulin and insulin. Aim 1. The ERK and PI3K pathways, activated by heregulin, are required for SC proliferation and cAMP regulates the kinetics of both pathways at unknown sites upstream of MEK and Akt, respectively. The target(s) of cAMP mediating the heregulin-dependent ERK and Akt activation in rat and human SCs will be identified. Specifically, the role of Rap1 and B-Raf in cAMP regulation of MEK and Akt will be determined. Aim 2. cAMP, in the presence of insulin, induces the expression of myelination-associated genes (MGs) in cultured SCs . Newer work suggests that neuregulin is also involved. The regulation of the expression of mRNA and protein for Krox-20, a master regulator of MG expression and myelination, by cAMP, neuregulin and insulin will be studied. Further, the role of the ERK, PI3K, PKA and EPAC pathways in these events, and the linkage between signaling and the activation of the Krox-20 promoter will be determined. Aim 3. The elevation of cAMP mimics axonal signals in the regulation of SC proliferation and myelination. To test if axonal contact increases cAMP levels in SC, fluorescent cAMP sensors or reporters will be used to determine changes in the level of cAMP after contact axons. These changes will be correlated with changes in the patterns of activation of ERK and Akt and with SC proliferation. Specific inhibitors of adenylyl cyclase, PKA, ERK and Akt will be used to define the path of cAMP-mediated signaling by axons. To study the role of cAMP in myelin formation, the change in cAMP levels following the addition of ascorbate, which precisely triggers myelination, will be monitored, and correlated to Krox-20 induction and MBP expression. Antagonist of PKA, ERK and Akt will be used to determine their roles in myelin formation. Aim 4. Studies in this laboratory (Pearse et al., Nat Med 10:610-6, 2004) have shown that cAMP dramatically increases myelination after SC transplantation. Elevated cAMP could exert its action on neurons in the host spinal cord or on the transplanted SC or both. To determine if cAMP-activated pathways in SCs are involved, cAMP levels and the response of intracellular targets of cAMP, will be monitored in the transplanted cells, and correlated to the arrival of regenerating axons and the onset of myelination. The completion of these experiments will improve our understanding of how cAMP regulates normal SC proliferation and myelination, specifically, and signaling from receptor tyrosine kinases in general.